anti-β2GPI IgG Research Articles

Up-regulation of adhesion molecule expression and induction of TNF-α on vascular endothelial cells by antibody against human parvovirus B19 VP1 unique region protein

Background Human parvovirus B19 infection has been frequently described as a cause or trigger of various autoimmune diseases. In previous studies, we have postulated the association among human parvovirus B19 (B19)-VP1 unique region (VP1u), production of anti-beta2-glycoprotein I (anti-β2GPI) antibody and anti-phospholipid syndrome (APS)-like autoimmunity. However, the precise role of B19-VP1u in induction of APS is still obscure. Methods To further elucidate the pathogenic roles of VP1u in B19 infection and autoimmunity, we examined the effect of anti-B19-VP1u IgG antibodies on endothelial cells that is recognized to play crucial roles in APS. Human vascular endothelial cells, ECV-304, were incubated with various preparations of purified human or rabbit IgG. The activation of endothelial cells and production of cytokines were assessed by flow cytometry and ELISA, respectively. Results Purified IgG from rabbits immunized with recombinant B19-VP1u proteins can up-regulate ICAM-1 (CD54), VCAM-1 (CD106), E-selectin (CD62E), MHC class II (HLA-DR, DP, DQ) molecule expression, and TNF-α production in endothelial cells as compared to those endothelial cells cultured with control IgG. Additionally, significantly increased phosphorylated-P38 mitogen-activated protein kinase (P38 MAPK) and iNOS were observed in both human anti-β2GPI IgG and rabbit anti-B19-VP1u IgG treated-ECV-304 cells, respectively. Conclusions These experimental results imply that antibodies against B19-VP1u play important roles in the immunopathological processes as well as human anti-β2GPI IgG that leads to development of APS by involving p38 phosphorylation and iNOS activation. It could provide a clue in understanding the role of anti-B19-VP1u antibodies in APS manifestations.

Intracellular trafficking of β 2-glycoprotein I complexes with lipid vesicles in macrophages: Implications on the development of antiphospholipid syndrome

β 2-Glycoprotein I (β 2GPI) is known as a major autoantigen for antiphospholipid antibodies. Our recent data show that binding of β 2GPI to oxidized low-density lipoprotein (oxLDL) or to liposomes containing anionic phospholipid(s) may facilitate the presentation of β 2GPI's epitope by macrophages/dendritic cells to autoreactive T cells. In the present study, we investigated intracellular trafficking of β 2GPI and its complexes with oxLDL or liposomes containing phosphatidylserine (PS-liposomes) in mouse macrophage-like J774 cells. A relatively small amount of non-complexed β 2GPI was taken up and stagnated in the late endosome after incubating for 16 h. In contrast, β 2GPI complexes with oxLDL or PS-liposomes were transported into the lysosome. In the presence of the IgG anti-β 2GPI autoantibody, WB-CAL-1, β 2GPI/oxLDL complexes were rapidly incorporated into intracellular space and were finally localized in the lysosome. Interestingly, in vitro pulses by β 2GPI/oxLDL complexes together with WB-CAL-1 led to the expression of membranous CD36 as well as Fcγ type I receptors (FcγRI). These observations suggest that IgG immune complexes of β 2GPI/oxLDL provide not only FcγRI- but also scavenger receptor-mediated uptake of β 2GPI/oxLDL complexes by macrophages. Thus, β 2GPI/oxLDL complexes as a major atherogenic autoantigen and IgG anti-β 2GPI autoantibodies may facilitate antigen presentation and foam cell formation in antiphospholipid syndrome.

Biosensor Analysis of β2-Glycoprotein I–Reactive Autoantibodies: Evidence for Isotype-Specific Binding and Differentiation of Pathogenic from Infection-Induced Antibodies

For the laboratory diagnosis of the antiphospholipid syndrome (APS) we developed a biosensor with the ability to distinguish between disease-relevant anti-beta2-glycoprotein I (beta2GPI) autoantibodies (anti-beta2GPI) and pathogen-specific beta2GPI cross-reactive antibodies that occur transiently during infections. We used a surface plasmon resonance (SPR) biosensor device. For the detection of anti-beta2GPI in serum samples, affinity-purified human beta2GPI was covalently attached to a functionalized n-alkanethiol self-assembling monolayer on the biosensor chip. After verifying the specificity of the biosensor system with a panel of monoclonal antibodies to beta2GPI, we analyzed sera from healthy donors and patients suffering from APS, systemic lupus erythematosus (SLE), syphilis, or parvovirus B19 infections. The SPR results were compared with beta2GPI-specific ELISA. Using the SPR biosensor, we recorded antigen binding curves with response levels in the range of 50-500, resonance units (RU) for anti-beta2GPI ELISA-positive APS patient sera. The amplitudes of the antiphospholipid antibody (APL) responses in the biosensor correlated with the overall IgG and IgM anti-beta2GPI ELISA titers with a correlation coefficient of 0.87. Moreover, we observed immunoglobulin isotype-specific association and dissociation profiles for APL binding of different APS patient sera to the biosensor-immobilized beta2GPI. In contrast to APS patient samples, no significant anti-beta2GPI binding (response levels <35 RU) was observed in samples from healthy individuals or from patients suffering from SLE, syphilis, or parvovirus B19 infection. The SPR biosensor system enables specific detection of APS-associated beta2GPI-reactive APL and differentiation from beta2GPI cross-reactive antibodies that occur frequently during acute infections. The established association/dissociation plot for anti-beta2GPI responses in APS patient sera gives additional information regarding the influence of anti-beta2GPI IgG and IgM isotype distribution.

Antibodies to β 2-glycoprotein-I: Relation of anticardiolipin antibodies with clinical and laboratory parameters in patients with systemic lupus erythematosus

Objectives: There are controversial reports on the frequency of antiphospholipid antibodies (aPL) in patients with systemic lupus erythematosus (SLE). Thus, we aimed to determine the frequency and clinical importance of aPL isotypes in Turkish patients with SLE. Design and methods: Fifty-nine patients with SLE and 41 healthy controls were included. Serum aPL levels were measured both in patients and healthy subjects by ELISA. Results: Fifteen of the patients with SLE had the antiphospholipid syndrome (APS) (25.4%). The percentage of anticardiolipin antibody (aCL)-positive SLE patients among all patients was 56%. At least one isotype of anti-β 2-glycoprotein I (β 2-GPI) antibody was positive in 83% of patients. The positivity rates of aCL and anti-β 2-GPI antibodies in patients with or without APS were higher than the healthy controls. There were positive correlations between isotypes of IgM aCL, IgG and IgM anti-β 2-GPI and manifestations of APS. Conclusion: It seems that the isotypes of IgM aCL, IgG and IgM anti-β 2-GPI are correlated with manifestations of APS. They may play a role in pathogenesis and may be helpful in establishing the diagnosis.

Patients with atherosclerotic syndrome, negative in anti-cardiolipin assays, make IgA autoantibodies that preferentially target domain 4 of β 2-GPI

Autoantibodies targeting β 2-glycoprotein l (β 2-GPI), a component of the atherosclerotic plaque, are commonly found in patients with acute ischemic syndromes. Serum samples from APS (antiphospholipid syndrome) patients and from cardiovascular patients exhibiting acute atherosclerotic syndromes were analyzed for IgG and IgA antibodies in both anti-β 2-GPI and anticardiolipin (aCL) ELISA assays. All of the APS samples used here were positive in both assays. Serum samples from 382 atherosclerosis patients were also analyzed for IgG and IgA antibodies in the same assays. In sharp contrast to the APS samples, we found that only 1% of the samples from atherosclerosis patients were positive for IgA aCL, and 1.6% positive for IgG aCL, whereas 35.6% were positive for IgA anti-β 2-GPI and only 1.6% for IgG anti-β 2-GPI. The antigenic specificity of 29 serum samples from atherosclerosis patients was evaluated. Six different recombinant domain-deleted mutants (DM) of human β 2-GPI and full-length human β 2-GPI (wild-type) were used in competitive inhibition assays to inhibit the autoantibodies from binding in the anti-β 2-GPI ELISA assays. Domain-deleted mutants D—345 and D---45 inhibited the binding in the IgA anti-β 2-GPI assay, suggesting that these autoantibodies recognize domain 4 of the β 2-GPI molecule. These results clearly show that IgA anti-β 2-GPI autoantibodies from atherosclerotic patients are distinct from IgA autoantibodies found in APS samples.

Antiphospholipid antibody ELISAs: Survey on the performance of clinical laboratories assessed by using lyophilized affinity-purified IgG with anticardiolipin and anti-β 2-Glycoprotein I activity

Lupus anticoagulant (LA) and anticardiolipin (aCL) antibodies are the classical tests used to diagnose the antiphospholipid syndrome (APS). Unfortunately, since these are nonspecific and standardization is lacking, the results of laboratory work-ups upon which diagnosis are made are often misleading. The performance of clinical laboratories in detecting LA using lyophilised affinity purified immunoglobulin has been previously reported. The same material was used to investigate the inter-laboratory variability of aCL and anti-β 2-Glycoprotein I (β 2-GPI) antibody measurements. Laboratories were asked to test normal plasma spiked with purified IgG or distilled water in order to obtain 3 samples positive for aCL and anti-β 2-GPI at different antibody concentration (A, B and C) and 3 samples of normal plasma. Thirty-five laboratories participated and interpreted their test results. All performed an ELISA for IgG aCL antibodies, while 17 also tested samples using IgG anti-β 2-GPI antibody ELISA. Sensitivity and specificity were calculated on the basis of the responses provided by each laboratory. Overall, 99/105 samples were correctly interpreted as positive and 97/101 as negative for the presence of IgG aCL, corresponding to a sensitivity and specificity of 94% and 96%, respectively. Likewise, 46/51 samples were correctly defined as positive and 50/51 as negative for the presence of IgG anti-β 2-GPI corresponding to a sensitivity and specificity of 90% and 98%, respectively. A wide variability in results pertaining to the positive samples was found for aCL-ELISA (coefficient of variation of 79%, 59%, and 53% for samples A, B, and C, respectively) as well as for aβ 2-GPI-ELISA (coefficient of variation of 85%, 95%, and 50% for samples A, B, and C, respectively). This was confirmed when the analysis was restricted to those centres using the same commercial kit. Median antibody concentrations reported by centres for positive samples were consistent with the prolongation of coagulation tests assessing lupus anticoagulant (LA). Among these, dRVVT showed a good sensitivity and linear correlation with aCL antibody concentration. In conclusion, on the whole this survey found correct interpretation of positive and negative samples by both ELISAs. Nonetheless the high variability of reported data remains a major problem that only a consensus on the part of laboratories and manufacturers to utilize standard, uniform materials and procedures can hope to overcome.

The role of β2-glycoprotein I (β 2GPI) in the activation of plasminogen

β2-glycoprotein I (β 2GPI) is a glycoprotein of unknown physiological function. It is the main target antigen for antiphospholipid antibodies in patients with antiphospholipid syndrome (APS). β 2GPI binds with high affinity to the atherogenic lipoprotein Lp(a) which shares structural homology with plasminogen, a key molecule in the fibrinolytic system. Impaired fibrinolysis has been described in APS. The present work reports the interaction between β 2GPI and Glu-Plasminogen which may explain the recently described proteolytic effect of plasmin on β 2GPI. In the process of Glu-Plasminogen activation, we found an increase in plasmin generation both at fibrin and cellular surface level as a function of the concentration of β 2GPI added, suggesting an important role as a cofactor in the trimolecular complex β 2GPI-Plasminogen-tPA. This phenomenon represents a novel regulatory step both in the positive feedback mechanism for extrinsic fibrinolysis and in antithrombotic regulation. IgG anti-β 2GPI antibodies recognized the β 2GPI at the endothelial surface inducing its activation with an increase of ICAM-I and a decrease in the expression of thrombomodulin favoring a pro-thrombotic state in the vascular endothelium. The interference in the plasmin conversion by anti-β 2GPI antibodies could generate thrombosis as observed in APS.

Antibodies Against β2‐Glycoprotein I Complexed With an Oxidised Lipoprotein Relate to Intima Thickening of Carotid Arteries in Primary Antiphospholipid Syndrome

To explore whether antibodies against β2-glycoprotein I (β2GPI) complexed to 7-ketocholesteryl-9-carboxynonanoate (oxLig-1) and to oxidised low-density lipoproteins (oxLDL) relate to paraoxonase activity (PONa) and/or intima media thickness (IMT) of carotid arteries in primary antiphospholipid syndrome (PAPS). As many as 29 thrombotic patients with PAPS, 10 subjects with idiopathic antiphospholipid antibodies (aPL) without thrombosis, 17 thrombotic patients with inherited thrombophilia and 23 healthy controls were investigated. The following were measured in all participants: β2GPI−oxLDL complexes, IgG anti-β2GPI−oxLig-1, IgG anti-β2GPI−oxLDL antibodies (ELISA), PONa, (para-nitrophenol method), IMT of common carotid (CC) artery, carotid bifurcation (B), internal carotid (IC) by high resolution sonography. β2GPI−oxLDL complex was highest in the control group (p < 0.01), whereas, IgG anti-β2GPI−oxLig1 and IgG anti-β2GPI−oxLDL were highest in PAPS (p < 0.0001). In healthy controls, β2GPI−oxLDL complexes positively correlated to IMT of the IC (p = 0.007) and negatively to PONa after correction for age (p < 0.03). PONa inversely correlated with age (p = 0.008). In PAPS, IgG anti-2GPI−oxLig-1 independently predicted PONa (p = 0.02) and IMT of B (p = 0.003), CC, (p = 0.03) and of IC (p = 0.04). In PAPS, PONa inversely correlated to the IMT of B, CC and IC (p = 0.01, 0.02 and 0.003, respectively). IgG anti-2GPI−oxLig-1 may be involved in PAPS related atherogenesis via decreased PON activity.

Anti-phospholipid antibodies following vaccination with recombinant hepatitis B vaccine

This study was undertaken to evaluate the possible role of hepatitis B recombinant vaccine inducing the synthesis of IgG and IgM anti-cardiolipin antibodies (aCL), antibodies against beta(2)GPI (anti-beta(2)GPI), lupus anti-coagulant (LA), anti-nuclear antibodies and antibodies against extractable nuclear antigens (anti-ENA). The study population consisted of 85 healthy students (63 female, 22 male; mean age 20.8 years), vaccinated with three doses of recombinant DNA hepatitis B vaccine. One month after vaccination with the first dose of hepatitis B vaccine a minority of vaccinated individuals showed changes in IgG or IgM aCL or anti-beta(2)GPI or LA activity (P < 0.001). Among subjects in whom changes of IgG anti-beta(2)GPI were observed, a significantly higher number of increased (8/85) than decreased (2/85) values were found (P < 0.01). Analyses of paired data showed that differences in aCL or anti-beta(2)GPI levels before vaccination or 1 month later did not reach statistical significance. In two people aCL transitorily reached medium positivity after the first dose of hepatitis B vaccine with a drop 5 months later. Similar evident anti-beta(2)GPI fluctuation was also observed in one person. Another participant was initially low positive for IgG anti-beta2GPI and the levels were increasing after vaccination. Two participants became positive for anti-nuclear antibodies during 6 months' follow-up. There were no sex-dependent differences in tested antibodies observed and no associations between levels of aPL and levels of anti-HBV antibodies. We conclude that HBV can induce aPL, although rarely. In genetically susceptible individuals or together with some other triggers such combination might confer the risk of developing a continuous autoimmune response in an individual.

β 2-glycoprotein I: antiphospholipid syndrome and T-cell reactivity

There is increasing evidence showing that recurrent thrombosis and intrauterine fetal loss in antiphospholipid syndrome (APS) are attributable to antiphospholipid (aPL) antibodies. We have recently identified autoreactive CD4 + T cells to β 2-glycoprotein I (β 2GPI) that promote production of pathogenic antiphospholipid antibodies. β 2GPI-specific CD4 + T cells preferentially recognize the antigenic peptide containing the major phospholipid (PL)-binding site in the context of DR53. T-cell helper activity that stimulates B cells to produce IgG anti-β 2GPI antibodies is mediated through IL-6 and CD40–CD154 interaction. β 2GPI-specific T cells respond to reduced β 2GPI and recombinant β 2GPI fragments produced in a bacterial expression system but not to native β 2GPI, indicating that the epitopes recognized by β 2GPI-specific T cells are ‘cryptic’ determinants, which are generated at a subthreshold level by the processing of native β 2GPI under normal circumstances. Although β 2GPI-specific T cells are detected in both APS patients and healthy individuals, these autoreactive T cells are activated in vivo in APS patients but not in healthy individuals. These findings indicate activation of β 2GPI-specific T cells and subsequent production of pathogenic anti-β 2GPI antibodies can be induced by the exposure of such T cells to cryptic peptides of β 2GPI efficiently presented by functional antigen-presenting cells (APC). Delineating the mechanisms that induce the efficient processing and presentation of cryptic determinants of β 2GPI as a consequence of antigen processing would clarify the etiology that initiates the autoantibody response in APS.

IgG autoantibodies against beta2-glycoprotein I complexed with a lipid ligand derived from oxidized low-density lipoprotein are associated with arterial thrombosis in antiphospholipid syndrome.

We recently reported [J. Lipid Res. 42 (2001), 697; 43 (2002), 1486; 44 (2003), 716] that β2-glycoprotein I (β2GPI) forms complexes with oxidized LDL (oxLDL) and autoantibodies against these complexes are present in patients with SLE and antiphospholipid syndrome (APS). The relationship of β2GPI/oxLDL complexes and IgG autoantibodies against β2GPI complexed with oxLig-1 (an oxLDL-derived ligand) with clinical manifestations of APS was studied in 150 APS and SLE patients. The β2GPI/oxLDL levels of APS patients were similar to those of SLE patients without APS, but they were significantly higher than healthy individuals. There was no difference in the complex levels among the patients with arterial, venous thrombosis, or pregnancy morbidity. IgG anti-β2GPI/oxLig-1 levels of APS were significantly higher than those of SLE without APS and healthy individuals. Further, antibody levels of APS patients with arterial thrombosis were significantly higher than those patients with venous thrombosis and pregnancy morbidity. Thus, oxidation of LDL leads the complex formation with β2GPI in SLE and APS patients. In contrast, anti-β2GPI/oxLig-1 autoantibodies were generated only in APS and were strongly associated with arterial thrombosis. These results suggest that autoantibodies against β2GPI/oxLDL complexes are etiologically important in the development of atherosclerosis in APS.

Antiphospholipid Antibodies in Patients with Lupus Nephritis

The aim of this study was to compare the prevalence of anticardiolipin antibodies with other types of antiphospholipid antibodies (aPL) (antiphosphatidylserine—aPS, antiphosphatidylinositol—aPI, antiphosphatidylethanolamine—aPE) in patients with lupus nephritis and to find if the examination of a panel of various aPL is valuable for further diagnosis of patients. Additionally we determined the levels of autoantibodies against β2-glycoprotein I (β2GPI) and oxidized low-density lipoprotein (anti-oxLDL) and also investigated the relationship between antibodies against β2GPI and oxLDL, which were assessed by ELISA methods. Twenty-two patients with lupus nephritis were studied. The control group consisted of 62 healthy blood donors. A statistically significant higher occurrence of all aPLs in the patients with lupus nephritis in comparison to the control group was found. The prevalence of polyspecific antibodies, which reacted with at least two various phospholipids, was 82% in the group of SLE patients. Significantly higher levels of IgG anti-β2GPI in the sera of SLE patients (p = 0.0003) was detected. The levels of anti-oxLDL in the sera of the patients group did not differ significantly from the control one. Some positive samples for anti-β2GPI and negative for aCL or anti-oxLDL and vice versa were found. It can be concluded that the production of aPL including anti-β2GPI and anti-oxLDL in the lupus nephritis patients is higher in comparison with healthy blood donors. We assume that the estimation of various types of aPL may be important in the selection of the group patients with renal diseases. The synthesis of aPL can reflect the spreading of the autoimmune response for several antigens modified on the vessel wall.

Anticardiolipin and Anti-β 2-Glycoprotein-I Antibodies in Hypertensive Disorders of Pregnancy

Background This study was undertaken in order to determine the frequency of anticardiolipin (aCL) and anti-β 2-glycoprotein-I antibodies (anti-β 2GP-I) in patients with hypertensive disorders of pregnancy. Methods We studied aCL (IgG and IgM) and IgG anti-β 2GP-I by ELISA in the serum and IgG aCL and anti-β 2GP-I in the urine of 125 women with hypertensive disorders of pregnancy (cases) (75 had preeclampsia, 25 had eclampsia, and 25 had chronic hypertension). One hundred and twenty seven normal women were used as controls. Antibody positivity was defined as above the 90 th percentile of the controls. Results We found no differences in frequency of serum anti-β 2GP-I or serum and urinary aCL between cases and controls. In contrast, we found that frequency of IgG anti-β 2GP-I was higher in the urine of cases (26.1%) compared to controls (9.4%, p = 0.001). Strength of association was stronger for urinary anti-β 2-glycoprotein-I in women with preeclampsia (odds ratio [OR] = 4.3, 95% confidence limit [CI 95%] = 1.95–9.62, p <0.0001). Cases and the subgroup of preeclamptic patients also had higher titers of urinary IgG anti-β 2GP-I than control women ( p <0.0001). Conclusions Our work suggests that urine testing is necessary in order to ascertain whether antibodies to β 2GP-I are associated with preeclampsia.

The inhibition of protein C anticoagulant activity by anti-β2-glycoprotein I (β2GPI) antibodies isolated from patients with antiphospholipid syndrome by chromatography methods

Antiphospholipid antibodies (aPL) are associated with an increased risk of thrombosis; however, the mechanism remains unknown. Recent studies have focused on the impediment of protein C anticoagulant activity by anti-β2-glycoprotein I (β2GPI) antibodies (aβ2GPI Ab). We purified IgG fractions containing a high concentration of aβ2GPI Ab from patients with antiphospholipid syndrome (APS) and then investigated the effect of purified aβ2GPI Ab on the activity of activated protein C (APC). Using a three-step chromatography method (DEAE-sepharose column, phosphatidylserine polyacrylamide gel column dependent on the presence of β2GPI, and protein G column chromatography), we successfully isolated anti-β2GPI IgG from nine patients with APS. Seven of nine samples inhibited APC activity in a concentration-dependent manner only in the presence of β2GPI, as observed by a chromogenic assay that was able to determine thrombin activity even in the presence of APC. The extent of APC inhibition by these fractions appeared to be related to aβ2GPI Ab titers of the purified IgG. However, the inhibitory effect of IgG from patients was not detected in the absence of β2GPI. IgG purified from three normal subjects did not affect APC activity. Herein, we show a useful method for the isolation of IgG containing a high concentration of aβ2GPI Ab. Moreover, the present findings indicate that inhibition by aβ2GPI Ab on APC anticoagulant activity could explain one of the mechanisms for the thrombotic state in APS.

Anti-β2-glycoprotein I Antibody-mediated Inhibition of Activated Protein C Requires Binding of β2-glycoprotein I to Phospholipids

To clarify the role(s) of anti-beta(2) GPI antibodies on thrombosis in anti-phospholipid antibody syndromes (APS), the effect of IgG from three patients on activated protein C (APC) was investigated using phospholipid vesicles and purified proteins. Two of the total IgG inhibited APC activity in the presence of beta(2)GPI, whereas the third IgG did not. In addition, one IgG inhibited APC activity without beta(2)GPI. Anti-beta(2)GPI IgG from the two inhibitory IgG preparations inhibited APC activity only in the presence of beta(2)GPI. Inhibition was suppressed partially by excess APC and almost completely by excess phospholipid vesicles. Cleaved beta(2)GPI, a non-phospholipid-binding form, did not support inhibitory activity, even though the anti-beta(2)GPI IgG bound to the cleaved molecule. This study confirms that anti-beta(2)GPI antibodies from APS patients inhibit APC activity, and demonstrates the requirement of phospholipid binding of beta(2)GPI for expression of the inhibitory activity of these antibodies.

Distinguishing Features of Anti-β2 Glycoprotein I Antibodies between Patients with Leprosy and the Antiphospholipid Syndrome

Anticardiolipin (ACA), anti-beta2 glycoprotein I (beta2GPI), and antiprothrombin antibodies of IgG and IgM classes were quantitated by enzyme-linked immunosorbent assays in 176 untreated leprosy patients across the histopathological spectrum. Positivity rates ranged from 21% (IgG ACA) to 30% (IgM anti-prothrombin) versus 4% in healthy controls (p <10(-2) to 10(-3)). Levels of IgM anti-beta2GPI and IgG ACA were significantly higher in lepromatous leprosy and multibacillary patient subgroups. IgG3 was the most common subclass reactive to both beta2GPI and prothrombin in selected high-titer leprosy sera, unlike antibodies from patients with the antiphospholipid syndrome (APS) largely restricted to IgG2. In leprosy patients, but not in the APS control group, there was no statistical correlation between ACA and anti-beta2GPI antibody levels. Likewise, a large fraction of anti-beta2GPI positive sera (36/45 and 28/44 for IgG and IgM, respectively) were unreactive in the standard ACA assay. Most assayed anti-beta2GPI antibodies from leprosy patients showed (i) ability to recognize both human and bovine beta2GPI immobilized on non-irradiated polystyrene plates, (ii) concentration-dependent inhibition of binding by cardiolipin, and (iii) relatively high avidity binding to fluid-phase beta2GPI, thereby differing from those found in APS. Finally, the location of the major epitopic region on the beta2GPI molecule targeted by autoantibodies was different in leprosy and APS, as assessed by direct binding to domain I- and V-deleted mutants and competition with the mouse monoclonal antibody 8C3, directed at domain I. Thus, leprosy-related antiphospholipid antibodies comprise persistent IgG and IgM anti-beta2GPI that differ from APS-related ones with respect to IgG subclass, avidity and epitope specificity, possibly reflecting distinct pathophysiological significance.

Heterogeneous behaviour of anti-beta2-glycoprotein I antibodies on different "high binding" microtiter plates

We recently identified anti-β2GPI antibodies in a high proportion of sera from children with atopic dermatitis (AD) and showed that these anti- β2GPI most probably recognise domain V of β2GPI by contrast to anti-β2GPI from patients with the anti-phospholipid syndrome (APS) which epitopes apparently reside in domain I or IV. The aim of the present study was to compare the binding of IgG anti-β2GPI in AD and APS on four representative commerciallyavailable types of high binding microtiter plates. Selected plates: Costar, Nunc, Linbro and Sumilon C. Randomly selected sera from 29 children with AD and sera from 43 SLE patients (24 with secondary APS) were tested by anti-β2GPI ELISA using affinity purified β2GPI. Assays were calibrated with the HCAL, chimeric anti-β2GPI monoclonal antibodies with human γ1 constant regions. The calibration curves for HCAL were practically the same on all four types of plates. Sera from 7/24 APS patients with medium or high anti-β2GPI levels showed similar binding properties on all four plates, while 3/24 sera expressed values either slightly above or below the cut-off points. On the other hand, anti-β2GPI from AD sera showed very simmilar binding on Costar, Nunc and Linbro plates, while only 3/13 positive sera with the highest values on these 3 types of plates expressed low positive values for IgG anti-β2GPI on Sumilon C plates (Table ​(Table1).1). Except for one serum (low positive on Linbro plate) all sera from SLE patients without APS were negative on all the plates. Table 1 Our results point to substantial differences in the binding to β2GPI coated on different microtiter plates by anti-β2GPI in AD (with signs of APS) but not by anti-β2GPI in APS. In contrast to the other plate types, Sumilon C plates coated with β2GPI bound only minimally antibodies from AD children. If such anti-β2GPI prove non-thrombogenic, we will be able to increase the specificity of detecting anti-β2GPI relevant for APS by the use of this type of microtiter plates. Alternatively, if both anti-β2GPI specificities prove thrombogenic, we will be able to increase the sensitivity of the assay system by the use of other less discriminatory types of plates. k, slope of linear regression plot and R2 - determination coefficient: both compared to Costar. N, number of IgG anti-β2GPI positive sera in the group. *P < 0.001 (significant difference when compared with Costar, Nunc or Linbro)

Phospholipid-Bound β2-Glycoprotein I Induces the Production of Anti-Phospholipid Antibodies

Apoptotic-cell-bound β2-glycoprotein I (β2GPI), but not apoptotic cells or β2GPI alone, can induce the production of anti-phospholipid (anti-PL) antibodies (Ab) in normal mice. Although it is presumed that β2GPI binds to anionic phospholipid (PL) exposed on the apoptotic cell membrane, the precise nature of this complex and its immunogenicity is unclear. To address these issues, we investigated the structure and immunogenicity of human β2GPI in the presence of different PL that may be expressed on the surface of apoptotic cells. BALB/c mice were immunized intravenously (iv) with β2GPI in the presence of cardiolipin (CL), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylcholine (PC), or PS/PC (25%/75%) vesicles. Cardiolipin+β2GPI induced the highest levels of anti-β2GPI and anti-CL IgG Ab and lupus anticoagulant (LA) activity, while β2GPI with PC or PS/PC vesicles produced no significant anti-PL Ab. PS+β2GPI was somewhat immunogenic, but less so than PG+β2GPI. β2GPI was immunogenic in the presence of native (CLN), but not hydrogenated (CLH), CL. Circular dichroism analysis demonstrated that the structure of β2GPI was altered specifically by interaction with CLN, but not other anionic PL, including CLH. Similarly, the structure of CLNwas affected by interaction with β2GPI, as detected by31P nuclear magnetic resonance. These findings demonstrate that β2GPI complexed with CLNis structurally altered, highly immunogenic, and induces the production of IgG anti-PL Ab. Furthermore, the structural modification and the generation of immunogenic epitopes on β2GPI upon interaction with CLNrequire the presence of unsaturated fatty acid chains, suggesting a role for oxidation in this process.

Effect of Anti-β2Glycoprotein I Lupus Anticoagulants on Fibrin Polymerization and Fibrinolysis

Anti-β2-Glycoprotein I (β2GPI) autoantibodies are the prominent laboratory feature of Hughes syndrome. By prolonging some coagulation tests in the presence of exogenous phospholipids (PL), they behave as classical Lupus Anticoagulants (LA). We investigated the effect of 3 affinity-purified anti-β2GPI IgG preparations from patients with Hughes syndrome on fibrin polymerization and fibrinolysis of normal plasma, measured by comparing the optical densities of assay mixtures in the presence of the autoantibodies or normal IgG. The presence of anti-β2GPI IgG in diluted Russell Viper Venom Time (dR VVT) assays, carried out using a PL dilution of 1:8 or 1:64, resulted in a delay in theonset of polymerization by 30-40 and 60-70 s, respectively. Fibrin polymerization was complete after 250 s for both anti-02GPI IgG and normal IgG. The inhibitory effect of the anti-02GPI antibodies was not observed in the presence of excess PL, as expected for LA. Anti-β2GPI IgG increased the plateau level of polymerization when dRVVT was performed in the presence of 1.5 nM recombinant tissue plasminogen activator, but did not impair the fibrinolytic process, which was almost complete after 250 min. The autoantibodies did not delay the onset of fibrin polymerization in tests carried out using recombinant tissue factor. On the contrary, the autoantibodies enhanced polymerization in prothrombin time assays, and accelerated it in tissue thromboplastin inhibition tests, with no effect on fibrinolysis. These data provide evidence that anti-β2GPI LA may act as either anticoagulants or procoagulants in different in vitro coagulation tests.

Standardized measurement of major immunoglobulin class (IgG, IgA, and IgM) antibodies to β2glycoprotein I in patients with antiphospholipid syndrome

Patients with the antiphospholipid syndrome (APS) have autoantibodies directed against epitopes on beta2 glycoprotein I (beta2GPI). We describe herein the performance characteristics of standardized enzyme-linked immunosorbent assays (ELISAs) for anti-beta2GPI of the three major immunoglobulin classes: IgG, IgA, and IgM. All three assays generated highly linear standard curves (5 points, r > or = 0.993 for each); precision was excellent both intra-assay and run-to-run, with coefficients of variation (CV) ranging from 2.3% to 6.6%. Values for IgG anti-beta2GPI correlated strongly with those obtained by an earlier method (r = 0.80, P< 0.0001). A study group consisting of 203 healthy subjects was used to generate percentile-based reference intervals for all three classes of anti-beta2GPI. APS subjects' anti-beta2GPI were found to differ significantly (P <0.0001 for each) from those of the control population. All three assays correlated well with beta2GPI-dependent anticardiolipin antibody (aCL) measurements; IgG: r = 0.94 (P <0.0001), for IgA: r = 0.82 (P<0.001) and for IgM: r = 0.84 (P<0.0001). We suggest that these ELISAs may provide valuable standardized measurements of IgG, IgA, and IgM anti-P2GPI.